CN1737137A

CN1737137A - Plasmid DNA large scale purification process

Info

Publication number: CN1737137A
Application number: CN 200510090721
Authority: CN
Inventors: 朱鸿飞; 孙怀昌; 张泉
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-08-15
Filing date: 2005-08-15
Publication date: 2006-02-22
Anticipated expiration: 2025-08-15
Also published as: CN1307302C

Abstract

The invention relates to a plasmid DNA large scale purification process, which comprises using 3M postassium acetate, hexadecyl trimethyl-ammonium bromide (CTAB) and TritonX-114 to purify the plasmid DNA, as a result, the host RNA, the host mixed proteins, the host enterotoxins and host genome DNA can be removed effectively.

Description

Plasmid DNA large scale purification process

Technical field

The present invention relates to plasmid DNA large scale purification process.

Background technology

Dna vaccination is a kind of new and effective vaccine that has just developed in recent years, its principal character is that exogenous gene cloning is gone into eukaryon expression plasmid, after recombinant plasmid vector imported body, plasmid exists with episomal form, and in host the effective expression target protein as antigen, thereby reach the prevention eqpidemic disease purpose.

Compare with existing vaccine, the superiority of dna vaccination mainly show as following some: the proteantigen of generation can be offered to MHC II and MHC I quasi-molecule, and respectively with CD ₄ ⁺And CD ₈ ⁺Combine, cause comprehensive immune protection response; Can produce good immunoprotection to born of the same parents entozoas such as tubercule bacillus, leishmanias; Validity period is 1 year, and strong immunizing power is arranged; The design of vaccine is relative simple with transformation, and the preparation of vaccine is also relatively easy; People and animals are not had toxic side effect, and the virulence of not having is returned unfavorable factors such as strong; There are not necrosis, the granuloma of injection site, can improve meat quality, increase the benefit.

Though dna vaccination has above-mentioned numerous advantage, its specification of quality is also very strict, and according to The World Health Organization's standard, the purge process of plasmid DNA can not be used animal derived enzyme (RNaseA and Proteinase K); Can not use organic solvents such as the deleterious phenol of people and animals, chloroforms; Avoid a large amount of organic reagents such as inflammable, explosive dehydrated alcohol and Virahol that use.Purified plasmid DNA purity requirement height is with reference to people doctor's judgement criteria, its OD ₂₆₀/ OD ₂₈₀Need between 1.75-1.85, detect less than RNA with 1% agarose gel electrophoresis; Dihomocinchonine acid detects proteic content in 0.001 μ g/ μ g plasmid DNA; The endotoxin content that tachypleus amebocyte lysate detects should be less than 0.01EU/ μ g plasmid DNA; The content of host genome should be less than 10ng/ μ g plasmid DNA; Supercoiled amount should be greater than 90%.

The purifying of plasmid DNA is difficulty relatively, and major cause is that its molecular weight is big, electric charge negativity and friability.The electronegativity of dna double spiral phosphoric acid skeleton is consistent with the electric charge of intracellular toxin and host's foreign protein, makes to become complicated separating between plasmid DNA molecule and these molecules; In addition, the easily broken fragility of long molecular chain dna molecular has increased difficulty also for large scale plasmid purification DNA.

The method of large scale plasmid purification DNA has cesium chloride density gradient centrifugation, affinity chromatography, molecular retention method and the polycation precipitator method etc. at present.Wherein the cesium chloride density gradient centrifugation plasmid DNA purification is classic methods, though can reach relevant criterion with the purified plasmid DNA of this method, purity is also quite high.But it must make by long-time high speed centrifugation under the situation of using ethidium bromide and cesium chloride, and not only consuming time, consumption power, contaminate environment, and expense costliness no longer have been fit to continue to use.

Along with the rise of pollution-free motion, the affinity chromatography plasmid DNA purification demonstrates prospect preferably, and that uses at present comprises cation-exchange chromatography, anion-exchange chromatography, hydrophobic chromatography or the like more.The principle of this method mainly is to utilize the electronegativity and the wetting ability of plasmid DNA, and the effect of purifying and cesium chloride density gradient centrifugation are approaching or low slightly.Though affinity chromatography does not have serious problems such as environmental pollution, it is consuming time and cost is expensive, and need carry out pre-treatment, thereby has limited its further use.

Though carry out the imagination of plasmid DNA purifying with the molecular retention technology good, but actual effect is not good, still is in conceptual phase at present, and it mainly is a difference of utilizing each component molecular weight in the cellular component that is extracted, control the aperture and the transmembrane pressure of film well, thereby reach the purpose of plasmid DNA purification.According to roundup, this technology need be carried out pre-treatment usually, and purification step is more, and purification effect only is medium.Simultaneously, because plasmid DNA mainly exists with form elongated and that have a negative superhelix structure, its molecular weight does not become complete dependency with the aperture of the film of its actual needs, so the molecular retention technology can not be applicable to the purifying of plasmid DNA fully.

Summary of the invention

The invention provides the large scale purification method of the suitable plasmid DNA of a kind of price.

DNA purification process of the present invention comprises and has used cetyl trimethylammonium bromide (CTAB), 3M Potassium ethanoate and TrionX-114.

Particularly, purification process of the present invention comprises the following steps:

The first step: the fermentation of gene engineering recombinant bacterium: from seed bank, take out a reorganization bacterial classification, after line on the resistant panel grows up to single bacterium colony, in containing corresponding antibiotic liquid nutrient medium, carry out enlarged culturing, and be inoculated among the 10L Fa Jiao Guan with 10% ratio, at 30% dissolved oxygen, pH is the intestinal bacteria that 7.0 condition bottom fermentation contains recombinant plasmid, during the fermentation, and with OD ₆₀₀Monitor the growth conditions of reorganization bacterium, stop fermentation during the later stage, centrifugal collection reorganization bacterium thalline when bacterium reaches logarithmic phase;

Second step: heavily add 5ml solution I (25mM/L Tris-Cl according to the wet bacterium of every gram, 10mM/L EDTA, 50mM/L glucose) amount adds the solution I thalline of will recombinating and suspends, add solution II (0.2M NaOH according to 1: 2: 1.5 ratio then, 1%SDS) and solution III (5mM/L potassium acetate, glacial acetic acid 11.5ml, H ₂O 28.5ml) carries out sex change and renaturation, with centrifugal 10 minutes of 6000g and collect supernatant, get the Virahol or the two volumes dehydrated alcohol precipitation plasmid DNA of 0.7 times of volume of aliquot adding, after sedimentary plasmid DNA is dissolved with TE, with 1% agarose gel electrophoresis in conjunction with λ Hind III Marker, quantitative with the gel analysis software analysis, and calculate the plasmid DNA total amount that contains in the collected supernatant;

The 3rd step: according to the plasmid DNA total amount, add 5% cetyl trimethylammonium bromide (CTAB), after stirring with 200 rev/mins rotating speeds, in 20 ℃ with the centrifugal 15min collecting precipitation of 8000g, abandon supernatant, precipitation with the dissolving of 3M liquor kalii acetici after, at 4 ℃ with the centrifugal 15min of 10000g, abandon precipitation, keep supernatant;

The 4th step: in supernatant, add the Virahol of 0.7 times of volume or the dehydrated alcohol of 2 times of volumes, 4 ℃ with the centrifugal 15min of 10000g, keep precipitation, abandon supernatant, at last with plasmid DNA with 75% alcohol washed twice, and be dissolved in and do not contain in the endotoxic distilled water;

The 5th step: in the plasmid lysate, add a certain amount of TritonX-114, thermal agitation is placed on hatches 10 minutes on ice, the solution that will contain TritonX-114 then is heated to 38～58 ℃ (in one embodiment of the invention, this temperature is 50 ℃), after 10 minutes with 10000g centrifugal 10 minutes, the supernatant that is obtained was the plasmid DNA solution of purifying.

Though use cetyl trimethylammonium bromide (CTAB) and 3M Potassium ethanoate in the prior art, the effect of this two kinds of reagent principle of work in purifying process of the present invention and performance is different fully.

CTAB is a kind of nontoxic, harmless cationic detergent.Use the CTAB of high density as the genomic extraction reagent of sea-tangle among the patent ZL01114195.6, not only different fully with principle of the present invention and effective object, and secondary solvents such as phenol, chloroform have wherein been used, the present invention then utilizes the characteristic that CTAB can the selective precipitation plasmid DNA molecule under lower concentration, from contain host genome DNA, RNA, foreign protein and endotoxic solution, optionally precipitate plasmid DNA, CTAB can also be as a kind of positively charged ion coating agent simultaneously, and part plays immunostimulant and DNA package action.

" molecular cloning experiment guide " (third edition, Science Press, in January, 2003) 32-34 page scheme 3 has been described the key step that the SDS alkaline lysis prepares plasmid DNA, wherein used the 3M Potassium ethanoate, main effect is to utilize its potassium ion displacement sodium ion, and the chromosomal DNA of formation insolubility, high molecular RNA and potassium ion/SDS/ protein/cell walls mixture, the present invention utilizes the 3M Potassium ethanoate can dissolve the characteristic of CTAB and the insoluble glue compound of DNA specifically, thereby discharges plasmid DNA specifically from the CTAB that adds before and the insoluble glue compound of DNA.

The present invention uses TrionX-114 to utilize it can not lose the characteristic of plasmid DNA specifically in conjunction with bacterial endotoxin, effectively removes bacterial endotoxin from purified plasmid DNA.

Purifying process described in the invention can replace various chromatography purification methods, and the purifying cost obviously reduces purified plasmid DNA OD ₂₆₀/ OD ₂₈₀Between 1.75-1.85, endotoxin content can not produce toxic side effect to animal body less than 0.05EU/ μ g plasmid DNA.

Purifying process described in the invention is suitable for various plasmid vectors, purified plasmid DNA not only is suitable for breadboard cell transfecting test and dna vaccination immunity test, be more suitable in the mass preparation of carrying out the required dna vaccination of animal immune prevention, for the application of dna vaccination on animal provides good platform.

Advantage of the present invention:

1, plasmid DNA purification process described in the invention does not use animal derived RNase A and Proteinase K and to phenol, the chloroform of human body and animal toxic side effect, in addition, reduced the use of inflammableness organic solvent dehydrated alcohol and Virahol to greatest extent.

2, all can reach by every index of the purified plasmid DNA of the present invention or near the quality standard of association area such as U.S.'s food and drug administration (FDA).

Description of drawings

Fig. 1: illustrated is the growth curve that contains recombinant plasmid pcDNAKCpG genetic engineering bacterium.

Fig. 2: illustrated is blending ratio between reorganization thalline and the solution I.

Fig. 3: the illustrated selective precipitation curve that is CTAB to plasmid DNA.

Fig. 4: illustrated is the RNA agarose gel electrophoresis detected result of plasmid purification pcDNAKCpG.

Fig. 5: illustrated is the detected result (dihomocinchonine acid system) of foreign protein content in the plasmid purification.

Fig. 6: illustrated is the electrophoresis result that is used to prepare the genomic DNA fragment of probe.

Fig. 7: illustrated is genomic dna Southern trace detected result residual in the plasmid DNA purification.

Embodiment

The extraction of embodiment 1, recombinant plasmid pcDNAKCpG and purifying

1, (insertion CpG oligonucleotide obtains in available from the plasmid pcDNA3.0 of Invitrogen company to contain recombinant plasmid pcDNAKCpG, detailed description is referring to CN1554751A) fermentation of gene engineering recombinant bacterium (host bacterium DH5 α is available from Invitrogen company): from seed bank, take out a bacterial classification, after line on the resistant panel grows up to single bacterium colony, carry out enlarged culturing, and be inoculated among the 10L Fa Jiao Guan with 10% ratio, with 30% dissolved oxygen, pH is that 7.0 condition is fermented and contained the recombination bacillus coli of recombinant plasmid pcDNAKCpG, treats OD ₆₀₀Reach at 30 o'clock and stop fermentation (Fig. 1), centrifugal collection reorganization thalline takes by weighing the wet bacterium of 400 grams.

2, heavily add 5ml solution I (25mM/L Tris-Cl according to the wet bacterium of every gram, 10mM/L EDTA, 50mM/L glucose) amount adds the thalline suspension (Fig. 2) of will recombinate of 2000ml solution I, add 4000ml solution II (0.2M NaOH according to 1: 2: 1.5 ratio then, 1%SDS) with 3000ml solution III (5mM/L potassium acetate, glacial acetic acid 11.5ml, H ₂O 28.5ml) carries out sex change and renaturation, see molecular cloning experiment guide (third edition, Science Press, in January, 2003) p1579-1560 for details.Collect supernatant with 6000g after centrifugal 10 minutes, get the 1ml supernatant and add two volumes dehydrated alcohol precipitation plasmid DNA, after sedimentary plasmid DNA is dissolved with 100 μ l TE, through 1% agarose gel electrophoresis, in conjunction with λ Hind III Marker, quantitative with the gel analysis software analysis, the plasmid DNA total amount that calculates in the supernatant to be contained is 300mg.

3, according to the total amount of plasmid DNA, add 150ml 5%CTAB (available from Amersco company) (Fig. 3), after stirring with 200 rev/mins rotating speeds, in 20 ℃ with the centrifugal 15min collecting precipitation of 8000g, abandon supernatant.Precipitation with the dissolving of 150ml 3M liquor kalii acetici after, 4 ℃ with the centrifugal 15min of 10000g, abandon precipitation, keep supernatant.

4, in the 100ml supernatant, add 70ml Virahol or 140ml dehydrated alcohol, 4 ℃ with the centrifugal 15min of 10000g, keep precipitation, abandon supernatant.At last, plasmid DNA with 75% alcohol washed twice, and is dissolved in 30ml and does not contain in the endotoxic distilled water.

5, adding 0.33ml TritonX-114 (available from Sigma company) in the plasmid lysate, to make its final concentration be 1%, the thermal agitation mixing is placed on hatches 10 minutes on ice, the plasmid solution that will contain 1%TritonX-114 then is heated to 50 ℃, after 10 minutes with 10000g centrifugal 10 minutes, the careful supernatant of drawing did not have in the endotoxic container in another in aseptic.

The effect of purified plasmid: on 1% agarose gel electrophoresis, detect less than RNA (Fig. 2) by the purified plasmid DNA of the present invention; Super spirial plasmid accounts for 88% (Fig. 2) of total plasmid DNA; Detect less than host bacterium foreign protein (Fig. 3) with BCA (dihomocinchonine acid) method; Host bacterium genomic dna is controlled at (Fig. 4) below 1%; Detect with tachypleus amebocyte lysate (available from Xiamen tachypleus amebocyte lysate demonstration plant company limited), behind the purifying in the plasmid endotoxic content be the 50EU/mg plasmid DNA.

Use the effect of CTAB plasmid DNA purification separately: though use the purified plasmid DNA of CTAB also to detect less than RNA separately at 1% agarose, but owing to do not use the 3M Potassium ethanoate, plasmid DNA is difficult to discharge from CTAB and the insoluble glue compound of DNA, therefore, the rate of recovery of plasmid has only 20%.In addition, use separately that endotoxic content does not obviously meet the pharmaceutical industries standard greater than the 10000EU/mg plasmid DNA in the purified plasmid DNA of CTAB.

Claims

1, a kind of purification process of plasmid DNA, this method comprises the following steps:

Second step: the amount that heavily adds the 5ml solution I according to the wet bacterium of every gram adds the solution I thalline of will recombinating and suspends, carry out sex change and renaturation according to 1: 2: 1.5 ratio adding solution II and solution III then, with centrifugal 10 minutes of 6000g and collect supernatant, get the Virahol or the two volumes dehydrated alcohol precipitation plasmid DNA of 0.7 times of volume of aliquot adding, after sedimentary plasmid DNA is dissolved with TE, with 1% agarose gel electrophoresis in conjunction with λ Hind III Marker, quantitative with the gel analysis software analysis, and calculate the plasmid DNA total amount that contains in the collected supernatant;

The 5th step: in the plasmid lysate, add a certain amount of TritonX-114, thermal agitation is placed on hatches 10 minutes on ice, the solution that will contain TritonX-114 then is heated to 38～58 ℃, after 10 minutes with 10000g centrifugal 10 minutes, the supernatant that is obtained was the plasmid DNA solution of purifying.

2, plasmid DNA purification process according to claim 1, super spirial plasmid accounts for the 80-90% of total plasmid DNA gross weight in the plasmid DNA of wherein said purifying.